Task 2.1 Evaluation of the genetic variation in captive meagre broodstocks (led by FCPCT). Preliminary studies have indicated that meagre broodstock in Europe, which were formed from juveniles obtained from mainly a single commercial hatchery, have reduced genetic variability as the juveniles originated from a small group of parents. Fin clips will be taken from individuals in broodstock groups held for aquaculture purposes; a minimum of 30 fish will be sampled from each of the 10 different broodstock groups from the participating RTD centres and SMEs. The collection of finclips will be undertaken also in commercial hatcheries that are not participating in DIVERSIFY. Already defined genetic markers will be applied and existing multiplex optimised and basic population genetics parameters (allelic richness, heterozygosity indices and inbreeding coefficients) will be estimated in order to describe the genetic status of captive broodstock and identify future needs. These needs will probably be that the genetic pool must be enlarged with the incorporation of wild stock or the mixing of existing captive stocks to ensure that the genetic variability of the base populations used in European aquaculture will enable the genetic selection of desirable production traits whilst avoiding problems associated with inbreeding.

Task 2.2 Development of protocols for paired crossing in spontaneous spawning (led by IRTA). Previous studies (IRTA) examining parentage contribution, indicated that 86% of spawns in communal spawning tanks were from only two parents, indicating that planned single paired crosses could be achieved by isolated pairs. A total of six pairs will be selected that are at the right post vitellogenic stage or have flowing milt and placed in spawning tanks. At weekly intervals (total of 6 weeks), the pairs will be induced to spawn with an injection of GnRHa. From the second week onwards when fish are manipulated to inject the females, the males will be transferred to a different tank in order to be crossed with a different female, producing a total of 36 families at the end of the experiment. Egg fecundity and quality (i.e., morphology, fertilisation, hatching and larval survival) will be assessed using 96-well microtiter plates. Mictrotiter plates are loaded individually with fertilized eggs and are maintained in a controlled temperature incubator until yolk-sack absorption. The plates are examined under a stereoscope every day, and embryonic development, hatching and larval survival are monitored.Task 2.3 Description of sperm characteristics and cryopreservation methods (led by IFREMER). Part of the state-of-the-art of in vitro fertilization protocols is to have good quality sperm available when the females ovulate and ova are stripped. At three time-points in the spawning season, sperm quality of 10 males will be assessed by (a) sperm concentration through image analysis, (b) sperm motility (% mobile cell, velocity, linearity of tracks) using the CASA plugin of ImageJ software, (c) sperm membrane integrity by eosin/nigrosin sperm viability test and (d) sperm energetic status using ATP measurement kits. Then chilled storage will be studied and cryopreservation will be adapted using a commercial diluent (Cryofish from Cryobiosystem, France). The conditions of sperm dilution in terms of volume, osmolality, pH and cryoprotectant type will be tested by comparison of sperm motility performance based on CASA analysis

Task 2.4 Development of in vitro fertilization methods for planned crosses (led by IRTA). The state-of-the-art method to obtain planned crosses to produce desired families is strip spawning of eggs and in vitro fertilisation. Ten females (five wild and five cultured fish) at the right post vitellogenic stage (Duncan et al., 2012) will be induced to ovulate at weekly intervals with a single injection of GnRHa. The females will be examined for ovulation at predetermined hours post injection, based on preliminary data from HCMR indicating that ovulation occurs after 32-36 h at 19°C. When obtained, ovulated eggs will be fertilised with fresh or stored sperm and egg quality parameters determined as above. A data set will be built up that will determine the interaction between, female stage at stimulation, time of response to hormone injection (ovulation) and egg quality to identify optimal stripping times after hormone injection.Task 2.5 Development of Single Nucleotide Polymorphisms (SNP) marker tools for the genetic characterization of fast and slow growers (led by HCMR). One of the principal bottlenecks to meagre production is variable growth rates, causing uncertainty in the prediction of total yield from each ongrowing cycle. Fast and predictable growth is an important and highly desired trait, which affects the profitability of food animal production, since feed costs account for the largest proportion of production costs. SNPs explain the greatest part of the genetic differences between individuals and are suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. Therefore, this task aims at using SNPs to identify markers and genes associated with genetic variation in growth through Next Generation Sequencing (NGS) of the whole transcriptome of 10 fish from different families and phenotypic size (of the same age) that will provide a data-set of over 100,000 SNPs. Putative SNPs identified by RNA-Seq as presumably associated with growth traits will be spotted on a SNP-array and will be initially validated by genotyping fish of the discovery panel (fast versus slow growing fish) using the TaqMan OpenArray technology. Fin clip samples and phenotypic growth data (weight and morphometric measures) will be obtained from a total of 800 individual meagre from tasks in WP 20 Grow out husbandry - meagre: 200 fast and 200 slow growing juveniles from a group of known families and 100 fast and 100 slow growers from each of two commercial scale cage experiments. These fish are going to be genotyped for validation of the growth-associated SNPs.